Washing with alkaline solutions in protein A purification improves physicochemical properties of monoclonal antibodies.

Protein A affinity chromatography has been widely used for both laboratory scale purification and commercial manufacturing of monoclonal antibodies and Fc-fusion proteins. Protein A purification is specific and efficient. However, there still remain several issues to be addressed, such as incomplete clearance of impurities including host cell proteins, DNA, aggregates, etc. In addition, the effects of wash buffers in protein A purification on the physicochemical characteristics of antibodies have yet to be fully understood. Here we found a new purification protocol for monoclonal antibodies that can improve physicochemical properties of monoclonal antibodies simply by inserting an additional wash step with a basic buffer after the capture step to the conventional protein A purification. The effects of the alkaline wash on monoclonal antibodies were investigated in terms of physicochemical characteristics, yields, and impurity clearance. The simple insertion of an alkaline wash step resulted in protection of antibodies from irreversible aggregation, reduction in free thiols and impurities, an improvement in colloidal and storage stability, and enhanced yields. This new procedure is widely applicable to protein A affinity chromatography of monoclonal antibodies.

Urinary liver-type fatty acid binding protein as a useful biomarker in chronic kidney disease.

BACKGROUND: We reported that urinary L-FABP reflected the progression of chronic kidney disease (CKD). This study is aimed to evaluate the clinical significance of urinary liver type fatty acid binding protein (L-FABP) as a biomarker for monitoring CKD. METHODS: Urinary L-FABP was measured using human L-FABP ELISA kit (CMIC.Co., Ltd., Tokyo, Japan). The relations between urinary L-FABP and clinical parameters were evaluated in non-diabetic CKD (n = 48) for a year. In order to evaluate the influence of serum L-FABP derived from liver upon urinary L-FABP, both serum and urinary L-FABP were simultaneously measured in patients with CKD (n = 73). RESULTS: For monitoring CKD, the cut-off value in urinary L-FABP was determined as 17.4 microg/g.cr. by using a receiver operating characteristics (ROC) curve. Renal function deteriorated significantly more in patients with 'high' urinary L-FABP (n = 36) than in those with 'low' L-FABP (n = 12). The decrease in creatinine clearance was accompanied by an increase in urinary L-FABP, but not in urinary protein. Serum L-FABP in patients with CKD was not correlated with urinary L-FABP. CONCLUSION: Urinary excretion of L-FABP increases with the deterioration of renal function. Serum L-FABP did not influence on urinary L-FABP. Urinary L-FABP may be a useful clinical biomarker for monitoring CKD.

Classification of chemical compounds by protein-compound docking for use in designing a focused library.

We developed a new method for the classification of chemical compounds and protein pockets and applied it to a random screening experiment for macrophage migration inhibitory factor (MIF). The principal component analysis (PCA) method was applied to the protein-compound interaction matrix, which was given by thorough docking calculations between a set of many protein pockets and chemical compounds. Each compound and protein pocket was depicted as a point in the PCA spaces of compounds and proteins, respectively. This method was applied to distinguish active compounds from negative compounds of MIF. A random screening experiment for MIF was performed, and our method revealed that the active compounds were localized in the PCA space of compounds, while the negative compounds showed a wide distribution. Furthermore, protein pockets, which bind similar compounds, were classified and were found to form a cluster in the PCA space.

PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum.

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a K(m) value of 0.65 microM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 microM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 microM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 microM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 microM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.

Clinical evaluation of urinary excretion of liver-type fatty acid-binding protein as a marker for the monitoring of chronic kidney disease: a multicenter trial.

To confirm the clinical usefulness of the measurement of urinary liver-type fatty acid-binding protein (L-FABP) in chronic kidney disease (CKD), we carried out a multicenter trial. Clinical markers were measured in patients with nondiabetic CKD (n = 48) every 1 to 2 months for a year. We divided patients retrospectively into progression (n = 32) and nonprogression (n = 16) groups on the basis of the rate of disease progression, then assessed several clinical markers. Initially creatinine clearance (Ccr) was similar in the 2 groups; however, the urinary L-FABP level was significantly higher in the former group than in the latter (111.5 vs 53 microg/g creatinine, P < .001). For the monitoring CKD, we set the cutoff values for urinary L-FABP and urinary protein at 17.4 microg/g creatinine and 1.0 g/g creatinine, respectively. Urinary L-FABP was more sensitive than urinary protein in predicting the progression of CKD (93.8% and 68.8%, respectively). However, urinary protein showed greater specificity than did urinary L-FABP (93.8% and 62.5%, respectively). Over time, the progression of CKD tended to correlate with changes in urinary L-FABP (r = - .32, P < .05), but not in urinary protein (r = .18, not significant). The dynamics of urinary protein differed from that of urinary L-FABP, which increased as Ccr declined. Urinary L-FABP is more sensitive than urinary protein in predicting the progression of CKD. Urinary excretion of L-FABP increases with the deterioration of kidney function. Urinary L-FABP is therefore a useful clinical marker in the monitoring of CKD.

Urinary excretion of fatty acid-binding protein reflects stress overload on the proximal tubules.

Urinary excretion of human liver-type fatty acid-binding protein (hL-FABP), which is expressed in human proximal tubules and engaged in free fatty acid (FFA) metabolism, was reported to reflect the clinical prognosis of chronic kidney disease. Here we have investigated the pathophysiological significance of hL-FABP in a model of protein overload nephropathy. Because L-FABP is not expressed in the wild-type mice, we generated hL-FABP chromosomal gene transgenic (Tg) mice. Tg mice were intraperitoneally injected with bovine serum albumin (BSA) replete with FFAs (r-BSA group) or FFA-depleted BSA (d-BSA group). The r-BSA group developed significantly more severe tubulointerstitial damage than did the d-BSA group. Renal expression of the hL-FABP gene was more up-regulated, and urinary excretion of hL-FABP was significantly higher, in the r-BSA group than in the d-BSA group. Furthermore, compared with their wild-type littermates injected with r-BSA, the number of infiltrated macrophages was significantly attenuated in Tg mice injected with it on day 28. In patients with kidney disease (n = 50), urinary hL-FABP was correlated with both urinary protein and the severity of tubulointerstitial injury. In conclusion, our experimental model suggests that urinary excretion of hL-FABP reflects stresses, such as urinary protein overload, on the proximal tubules. The clinical observations support this hypothesis.

Urinary fatty acid-binding protein as a new clinical marker of the progression of chronic renal disease.

Previous studies have indicated that in massive proteinuria, free fatty acids (FFAs) bound to albumin were overloaded in the proximal tubule and exacerbated tubulointerstitial damage. Liver-type fatty acid-binding protein (L-FABP) is an intracellular carrier protein of FFAs that is expressed in the proximal tubule of human kidney. We sought to evaluate urinary L-FABP as a clinical marker in chronic renal disease. Urinary L-FABP was measured in patients with nondiabetic chronic renal disease (n = 120) with the use of a newly established ELISA method. We then monitored these patients for 15 to 51 months. Clinical data were analyzed with multivariate analysis. Urinary L-FABP was correlated with urinary protein, urinary alpha(1)-microglobulin, and serum creatinine concentrations. Urinary L-FABP at the start of follow-up (F = 17.1, r =.36, P <.0001) was selected as a significant clinical factor correlated with the progression rate, defined as a slope of a reciprocal of serum creatinine over time. We next selected the patients with mild renal dysfunction (n = 35) from all 120 patients and divided them into 2 groups according to progression rate: the progression group (n = 22) and the nonprogression group (n = 13). Serum creatinine and urinary protein concentrations and blood pressure at the start of follow-up were higher in the progression group than in the nonprogression group, although we detected no significant difference between the 2 groups. Urinary L-FABP was significantly higher in the former group than in the latter (P <.05). The results showed that urinary L-FABP reflected the clinical prognosis of chronic renal disease. Urinary L-FABP may be a clinical marker that can help predict the progression of chronic glomerular disease.

Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage.

BACKGROUND: Evidence indicates that urinary protein is associated with tubulointerstitial damage and thus it is an aggravating factor for chronic renal disease. As free fatty acids (FFAs) are bound to serum albumin, we hypothesized that FFAs were overloaded to the proximal tubule in massive proteinuria and thus caused tubulointerstitial damage. To test this hypothesis, massive proteinuria was provoked in mice and the renal damage examined. METHODS: Mice were intraperitoneally injected with bovine serum albumin (BSA) replete with FFAs (r-BSA group, N = 10), FFA-depleted BSA (d-BSA group, N = 10), or saline (saline group, N = 9) for 14 days. RESULTS: The kidneys of the r-BSA group showed severe tubulointerstitial damage and those of the d-BSA group showed mild tubulointerstitial damage. Urinary excretion of both total protein and mouse albumin were significantly higher in the r-BSA group than in the d-BSA group. To examine the proximal tubular uptake of albumin, the BSA content in the cultured mouse proximal tubules was measured by ELISA after 90 minutes of incubation with each BSA. In terms of the BSA content in the proximal tubules, there was no significant difference between the r-BSA and the d-BSA groups. These results indicate that r-BSA and d-BSA were similarly reabsorbed into the proximal tubule and that r-BSA causes severe tubulointerstitial damage. CONCLUSIONS: It is the FFAs bound to albumin, rather than albumin itself, which cause severe tubulointerstitial damage by being reabsorbed into the proximal tubule. To our knowledge, this is the first in vivo observation in which FFAs have caused severe tubulointerstitial injury.

Integrative control of rectoanal reflex in guinea pigs through lumbar colonic nerves.

The aim of the present study was to analyze the neuromodulation of rectoanal reflex activity by lumbar sympathetic nerves in guinea pigs. The mechanical activities of the rectum were recorded with a balloon connected to a pressure transducer, and those of the internal anal sphincter (IAS) were recorded with a custom-made strain gauge force transducer. Gradual and sustained rectal distension evoked the rectoanal reflex, causing cholinergic contractions of the rectum and synchronous nitrergic relaxations of the IAS. Section of the lumbar colonic nerves enhanced both rectal contractions and IAS relaxations. Section of the 13th thoracic cord abolished both rectal contractions and IAS relaxations, but section of the lumbar colonic nerves restored them. Lumbar sympathectomy and pithing sacral cords greatly diminished these rectal contractions and IAS relaxations, but the intrinsic reflex component remained. NG-nitro-L-arginine methyl ester enhanced the intrinsic reflex-mediated contraction of the rectum and abolished reflex-mediated relaxation of the IAS and converted into cholinergic contractions. The present results indicate that the extrinsic lumbar inhibitory outflow causes marked inhibition of the rectoanal reflex via the lumbar colonic nerves.